Cocultures are attractive microcosms for studying metabolic interactions in a controlled system. Cocultures involving diazotrophs, or organisms capable of reducing atmospheric nitrogen into ammonia, are of particular interest sustainable alternatives to the Haber-Bosch process a source of nitrogen fertilizer. While diazotrophs like Azotobacter vinelandii have successfully been engineered to produce ammonium aerobically, [1] these strains also exhibit a high substrate demand and overall inefficient carbon metabolism. Indeed, we have previously shown that the majority (88-90%) of the carbon consumed by A. vinelandii is spent on respiration, ultimately to protect its nitrogenase from oxygen, rather than ATP production for fueling nitrogen fixation and other cellular processes. Thus, a major challenge in engineering stable cocultures with nitrogen-fixers is to identify partners that can meet the high substrate demands of these diazotrophs while receiving limited nitrogen in return. We investigated the performance of four, fully nitrogen self-sufficient cocultures involving an ammonium-secreting strain of A. vinelandii paired with both wild-type and various engineered E. coli strains systematically chosen to model interactions ranging from commensalism to mutualism. 13C-metabolic flux analysis (MFA) of these cocultures unexpectedly revealed that A. vinelandii shares the majority of its fixed nitrogen. For example, even in commensalist pairings, E. coli constituted more than 60-75% of an exponentially growing coculture population, up to a total OD of ~5. Furthermore, using 13C-MFA, we identified additional cross-fed nutrients that are not otherwise secreted by either organism when grown in isolation. Finally, in preliminary evolution studies, we were able to isolate a clone of one of our engineered E. coli strains that had gained the ability to continue producing glucose for A. vinelandii even under complete nitrogen starvation. Ongoing work harnesses further adaptive evolution to not only increase cross-feeding, but also identify additional traits that enhance symbiosis.

Engineering cooperation in nitrogen self-sufficient cocultures of Azotobacter vinelandii and Escherichia coli

2019 Spring National Meeting

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